Antigen

Antigen Class

TFs and others

Antigen

Satb1

Cell type

Cell type Class

Neural

Cell type

Brain

MeSH Description

The part of CENTRAL NERVOUS SYSTEM that is contained within the skull (CRANIUM). Arising from the NEURAL TUBE, the embryonic brain is comprised of three major parts including PROSENCEPHALON (the forebrain); MESENCEPHALON (the midbrain); and RHOMBENCEPHALON (the hindbrain). The developed brain consists of CEREBRUM; CEREBELLUM; and other structures in the BRAIN STEM.

Sample

source_name

SATB1 Wild-type

cell type

Brain

age

5 weeks

strain

C57BL/6

antibody

Satb1 1583D

chip antibody vendor

home made

genotype

SATB1 Wild-type

chromatin

urea purified chromatin

Sequenced DNA Library

library_name

GSM5740048

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

chromatin preparation for urea ChIPseq experiment: The protocol for urea ChIP-seq is briefly described. Single cell suspension prepared from tissues were cross-linked with 1% formaldehyde for 10 min at room temperature. After quenching cross-linking reaction (0.125M Gly at final), these cells were lysed in buffer (4% SDS, 50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 100 mM NaCl) with Protease inhibitor (Roche) and RNase inhibitor and loaded on top of 8 M urea followed by ultracentrifugation in a Beckman TL-100 Ultracentrifuge for 4-5 hrs at 45K rpm (181,000g, g=relative centrifugation force or RCF; TLS55 rotor). Recovered chromatin pellet was sonicated using Branson sonifier (20% power, 10 sec on and 30 sec off for 4-6 cycles) in sonication buffer (0.5% Na-laurylsarcosine, 0.1% DOC, 1mM EDTA, 0.5mM EGTA, 10mM Tris-HCl, 100mM NaCl) with Protease inhibitor and RNase inhibitor. The reverse cross-linking of chromatin aliquot was done at 65°C overnight in buffer containing 1% SDS, 100 mM NaCl, 1 mM EDTA, 10mMTris-HCl. Samples were further treated by 100 µg/ml RNase A at 37°C for 1 hr, 200 µg/ml proteinase K at 60°C for more than 3 hrs, followed by DNA purification. For chromatin-immunoprecipitation, 1 µg of an antibody of choice were added to 10 ~ 20 µg of sonicated solubilized chromatin in 350 µl in dilution buffer (1% Triton X-100, 0.01% SDS, 1 mM EDTA, 15 mM Tris-HCl, 100 mM NaCl) and incubated overnight at 4°C. The chromatin fragments bound to antibodies (after incubation overnight) were captured using 40 µl of protein-A or protein-G Dynabeads (Invitrogen; pre-washed with 0.1% BSA [Fraction V, Sigma), by 4 hrs incubation time. After beads washing and DNA elution from beads, ChIP DNAs was reverse-crosslinked at 65oC overnight, followed by RNase-A treatment and proteinase K treatments as described above. The recovered DNA was further purified with a QIAquick PCR purification kit (Qiagen), with elution in 15µl of Qiagen Elution Buffer (alternatively, Phenol-chloroform extraction and ethanol precipitation). The purified DNAs were subjected to ChIP-seq library construction.

Sequencing Platform

instrument_model

Illumina HiSeq 4000

mm10

Number of total reads

48167715

Reads aligned (%)

69.1

Duplicates removed (%)

48.7

Number of peaks

57076 (qval < 1E-05)

mm9

Number of total reads

48167715

Reads aligned (%)

69.0

Duplicates removed (%)

48.8

Number of peaks

56909 (qval < 1E-05)